Test device and method for its use

ABSTRACT

A test device for determining a characteristic of a sample, particularly for determining substances in fluid samples. The device comprises a strip element which is composed of a material capable of transporting a developing fluid therealong by capillarity and which has a portion at a predetermined location on the strip element for receiving the test sample and portions at predetermined locations on the strip element incorporated with reagent means for providing a detectable response sensitive to the characteristic under determination. The beginning end portion of the strip element is immersed in the developing fluid which, as a result, traverses the length on the strip element, thereby promoting appropriate contact between the test sample and the reagent means resulting in the disposition of a detectable response at a predetermined location on the strip element, which response is a function of the characteristic under determination. The test device is particularly suited for performing binding assays, in particular those wherein a radioisotope is used as a label, such as radioimmunoassays.

This is a continuation of application Ser. No. 3,076, filed Jan. 12,1979, and which issued Nov. 25, 1980, as U.S. Pat. No. 4,235,601, whichis a continuation of application Ser. No. 887,886 filed Mar. 20, 1978,and now abandoned, which is a division of application Ser. No. 701,762,which was filed July 2, 1976, and which issued June 13, 1978, as U.S.Pat. No. 4,094,647.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to test devices and methods for their use indetecting a characteristic of a sample. In particular, the presentinvention relates to test devices and methods for their use inperforming binding assays such as for determining a ligand in or theliquid binding capacity of a fluid sample, such as a sample of abiological liquid. In preferred embodiment, the present inventionrelates to test devices for performing binding assays in which aradioisotope is used as a label.

2. Brief Description of the Prior Art

Test devices in the form of test strips have become commonplace in theanalysis of various types of samples, such as liquid samples in thenature of industrial fluids, biological fluids, and so forth, because ofthe convenience and speed of their use. Test strips designed fordetecting various clinically significant substances in biologicalfluids, such as urine and serum, in particular have been found to bevery advantageous in assisting the diagnosis and treatment of diseasedstates in man and other animals.

Conventional test strips generally comprise an absorbent or porousmatrix incorporated with indicator reagents, usually of a colorimetrictype. The sample to be tested is contacted with the reagent matrix, suchas by momentary immersion where the sample is liquid, and the indicatorresponse is observed after a set period of time. Such test strips havethe limitation that where more than one chemical reaction is involved,all of the test reactions must be mutually compatible since they alloccur in the presence of each other in the reagent matrix.

Recently, certain test strips have been developed which allow severaltest reactions to take place in a predetermined sequence. U.S. Pat. No.3,011,874 discloses a test strip representative of this type. Thedisclosed strip comprises a paper strip partitioned widthwise intovarious bands, first a blank band for immersion in a liquid test sample,followed successively by a reaction band, a gas liberation band, abarrier band, and an indicator band. However, there still exists along-felt and recognized need for further improvement.

First of all, the conventional test strips have limited sensitivity.Because the detectable response produced by the conventional strips isalmost always a color change, limited color resolution of the eye, andspectrometers as well, does not allow conventional test strips to detectsubstances, such as hormones, vitamins, and the like, which appear inbody fluids at concentrations below 0.1 mg/ml. Secondly, a relativelylarge sample volume must be provided in order to wet the entire reagentmatrix of the conventional test strips. The use of the test stripdescribed in the aforementioned U.S. Pat. No. 3,011,874 requires asample volume sufficient to wet by capillary absorption all of the stripup to the barrier band.

The known analytical methods for detecting substances which occur insamples in small amounts are based on the binding affinity of suchsubstances for certain synthetic or naturally produced binding agents.The most commonly used binding assay at the present time is probably theradioimmunoassay in which the substance to be determined competes undercontrolled conditions with a radiolabeled form of itself for binding toa limited quantity of specific antibody. The proportion of theradiolabeled form that successfully binds to antibody to that whichremains in a free state is a function of the amount of the substanceunder determination in the test sample. Because binding assay methodsrequire accurate and timed addition of minute quantities of reagents,the state of the art is that of time consuming and burdensome wetchemistry. The association of binding assay techniques with test deviceswhich attempt to simplify and reduce the cost of such assays has beenextremely limited despite the fact that many hundreds of technicalpapers have appeared in the literature over the past two decadesrelating to radioimmunoassay approaches alone.

U.S. Pat. No. 3,888,629 describes a device wherein the binding reactiontakes place in a disc-shaped matrix pad held in a section of a column.Free and bound label are separated by fitting a wash reservoir columnsection above the pad containing section and a column section containingan absorbent material below the pad containing section. As the washsolution is drawn through the reaction pad, free label is carried alongby the wash leaving the bound label behind because of the filtrationproperties of the reaction pad. This device would be cumbersome andawkward to use and requires several time consuming manipulative steps.

German Offenlegungsschrift No. 2,241,646 discloses a complex automatedinstrument for performing radioimmunoassays wherein the bindingreagents, i.e. the label and specific antibody, and an aliquot of theliquid to be tested are dispensed onto a cellulose strip at discretelocations. After an incubation period, the test areas on the strip arewashed by drawing a liquid therethrough by suction, thereby removingfree label. The level of radioactivity remaining at each test area isthen measured and related to the amount of unknown in the sample. Inaddition to requiring the use of an expensive and complex instrument,this method requires controlled dispensing of reagents and timing ofincubations, the same as the conventional wet chemistry methods.

It is therefore an object of the present invention to provide a noveltest device capable of application to analytical methods wherein a setof sequential test reactions is involved and wherein a minute samplesize may be used.

It is a further object of the present invention to provide a test devicecapable of application to analytical methods having sensitivities below0.1 mg/ml.

It is a particular object of the present invention to provide a testdevice useful in performing binding assays to detect characteristics offluid samples wherein the user is not required to dispense any of thebinding reagents or to carefully time incubation periods andparticularly wherein radioactive labels are employed, such as inradioimmunoassays.

SUMMARY OF THE INVENTION

The present invention provides a test device which satisfies theaforesaid objectives, such device comprising an elongated strip elementcomposed of a material capable of transporting a developing fluidlongitudinally therealong by capillarity. One of the end portions of thestrip element is designated the beginning end portion, and the other,the terminal end portion, to reflect the fact that the travel of thedeveloping fluid along the strip element begins by immersion or othercontact of the developing fluid with the beginning end portion and suchtravel terminates when the leading front of the developing fluid reachesthe terminal end portion. The strip element has a portion designated ormarked for receiving the sample to be tested and is incorporated at oneor more portions with reagent means comprising one or more reagentconstituents. All of the constituents of the reagent means may beincorporated with a single discrete portion of the strip element, someof the reagent constituents may be incorporated in combination with asingle discrete portion of the strip element with the remaining reagentconstituents being incorported, singularly or in any appropriatecombination, with one or more discrete portions of the strip element, oreach of the reagent constituents may be incorporated with separateportions of the strip element. The sequence of the various reagentconstituent incorporated portions of the strip element and the samplereceiving portion thereof, as well as the spacing therebetween, isestablished such that upon complete traversal of the strip element bythe developing fluid, a detectable response which is a function of thecharacteristic under determination is disposed at a predeterminedmeasuring location on the strip element.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1, 3, and 5, respectively, are front plan views of three differentforms of the test device of the present invention.

FIG. 2 is a cross-sectional view of the test device shown in FIG. 1taken along line 2--2.

FIG. 4 is a cross-sectional view of the test device shown in FIG. 3taken along line 4--4.

FIG. 6 is a rear plan view of the test device depicted in FIG. 5.

FIG. 7 is a cross-sectional view of the test device shown in FIG. 5taken along line 7--7.

FIG. 8 is a perspective view of an assembly comprising the test devicedepicted in FIGS. 5-7 enclosed in a sealed chamber with the device incontact with a volume of developing fluid.

FIG. 9 is a perspective view in partial cross-section of the assemblydepicted in FIG. 8 inverted and positioned in a well of an instrumentfor measuring the response of the reagent means.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring to the drawing, FIGS. 1 and 2 depict a test device 10comprising a strip element 11 composed of a material, usually bibulouspaper, which is absorbent relative to a selected developing fluid,usually an aqueous solution. Strip element 11 has a beginning endportion 12 and a terminal end portion 16. A sample receiving portion 13is designated on strip element 11 by appropriate marking means such as adried spot of a dye solution. Portions 14 and 15 of strip element 11 areincorporated with appropriate constituents of a reagent means selectedfor detecting a particular characteristic of a test sample. In use, thesample to be tested is dispensed on sample receiving portion 13 of stripelement 11 and beginning end portion 12 is immersed in the developingfluid which then begins to advance along strip element 11 by capillaritytowards terminal end portion 16. The developing fluid is selected sothat the dispensed sample and the constituents of the reagent means areappropriately combined as the developing fluid traverses strip element11. When the leading front of the developing fluid reaches terminal endportion 16, a detectable response of the reagent means which is relatedto the characteristic to be determined is disposed at a predeterminedmeasuring location on strip element 11, such as at portion 15. Suchresponse is then measured by appropriate means, for example, bymeasuring a chemical or physical property of such portion. For instance,if the detectable response of the reagent means is a physical propertysuch as fluorescence, light absorbance, or radioactivity, that propertycan be measured on the intact strip element. Also, if the detectableresponse is a chemical property, such as the appearance of a chemicalproduct or the disappearance of a chemical reactant, such property canbe measured by adding an indicator to the measuring location on thestrip element or by first separating the measuring location from theremainder of the strip element and making appropriate measurementsand/or reagent additions. Where the response is measured on the intactstrip element, for example at portion 15, it is useful to appropriatelymask all of the remaining surface of the strip element to assure thatthe measured response is only that associated with the measuringlocation, i.e. portion 15. As masking agent may be used, a shroud whichis opaque to the physical property being measured and which has anopening for registry with portion 15. Preferably an indicator meansresponsive to contact with the developing fluid is incorporated withterminal end portion 16 to signal completion of the test. It is alsopreferred that the volume of developing fluid into which beginning endportion 12 is immersed be equal to the precise volume of developingfluid that is taken up by strip element 11 upon arrival of the leadingedge of the developing fluid at terminal end portion 16, therebyresulting in the automatic termination of the transport of thedeveloping fluid at the proper time.

In one embodiment of the test device of the present invention fordetermining a ligand in a fluid sample, the reagent means comprisesappropriate binding reagents including a specific binding partner, suchas an antibody or other binding protein, for the ligand and a labelcomprising a labeled form of the ligand or of a binding analog thereof.Predetermined quantities of the label and the specific binding partner,the latter in an immobilized form, are incorporated with portions 14 and15 respectively. In use, as the developing fluid advances along stripelement 11, the sample and the label are mixed and transported intocontact with the immobilized binding partner whereupon the label and anyligand in the sample compete for binding to the binding partners. Thefraction of the label which successfully becomes bound to the bindingpartners thereby becomes immobilized at portion 15. As the developingfluid advances to terminal end portion 16, the unbound, or free,fraction of the label is transported a distance away from portion 15.The amount of label immobilized at portion 15 is then measuredappropriately and is related to the amount of ligand in the test sample.It is particularly useful to use as the label a radioactive form of theligand or of a binding analog thereof. If the radioactive label is aniodine isotope, an additional advantage of the test device results inthat contamination in the form of radioactive free iodide does notinterfere with measurements at portion 15 since such is transported adistance away from portion 15 by the advance of the developing fluid.

A variation of the test device described in the next preceedingparagraph is illustrated in FIGS. 3 and 4 of the drawing. Test device 20comprises a strip element 21 affixed to an inert support strip 22,usually made of a semi-rigid plastic, and having a beginning end portion23, a terminal end portion 28, and a sample receiving portion 25, whichin this embodiment is situated between reagent means incorporatedportions 24 and 26. Portion 24 of the strip element is incorporated withthe label and portion 26 is incorporated with the specific bindingpartner, which in this embodiment is not immobilized relative to thedeveloping fluid but rather is transportable thereby. In use, the sampleand label are mixed by the advancing developing fluid and competitivebinding for the binding partner occurs at portion 26. As the developingfluid advances farther, the resulting label-binding partner complexesare transported along strip element 21 along with the sample and freelabel, however, such complexes advance at a slower rate than that atwhich free label is carried. When the leading front of the developingfluid reaches terminal end portion 28, the label-binding partnercomplexes are disposed at portion 27 while free label has been carriedfarther towards terminal end portion 28. Measurement can then be made atportion 27 of strip element 21. Alternatively, portion 27 can beincorporated with an immobilized agent for the label-binding partnercomplexes, such as a second antibody or a protein precipitating agent,in order to assure localization of such complexes at portion 27.

A preferred form of the test device of the present invention isillustrated in FIGS. 5-9 of the drawing. Test device 30 comprises astrip element 51 folded widthwise over one end of an inert support strip32 in such a manner that the transverse edge of beginning end portion 33of strip element 31 is about even with the other edge of support 32 andthe transverse edge of terminal end portion 37 of strip element 31 isspaced a distance short of the same edge of support 32. Sample receivingportion 34 and reagent means incorporated portions 35 and 36 correspondto portions 13, 14, and 15, respectively, of test device 10 depicted inFIGS. 1 and 2. For example, for using test device 30 to detect a ligandin a liquid sample, portion 35 may be incorporated with a label andportion 36 with an immobilized binding partner. In this embodiment, testdevice 30 functions in a similar manner to test device 10 with thepredetermined measuring location being disposed at portion 36.

FIG. 8 illustrates an operative mode of a preferred test means 40 of thepresent invention. After the test sample has been dispensed onto samplereceiving portion 34 of the strip element 31, test device 30 isinserted, with beginning end portion 33 down, into test tube 41 whichcontains a volume of a developing fluid 42. The size of test tube 41 andthe dimensions of strip element 31 are selected preferably so that thevolume of developing fluid 42 is precisely the amount that is taken upby strip element 31 upon arrival of the leading edge of developing fluid42 at terminal end portion 37 and so that terminal end portion 37remains out of contact with developing fluid 42 when test device 30 isinitially inserted into tube 41. After sample inoculated test device 30is inserted into tube 41, friction cap 43 is fitted onto tube 41 to forma sealed chamber to prevent evaporation of developing fluid 42 duringthe traversal thereof along strip element 31.

FIG. 9 depicts a preferred manner of measuring the reagent meansresponse at portion 36 of strip element 31. It can be seen that bypositioning sample receiving portion 34 and reagent means incorporatedportions 35 and 36 appropriately on strip element 31 so that themeasuring location is at or proximate to the fold in strip element 31,measurement of the reagent means response can be convenientlyaccomplished by masking all of strip element 31 other than the portionproximate to the fold of strip element 31. Test means 40 is insertedinto shroud 50 which is opaque to the physical property characterizingthe response of the reagent means. The resulting assembly is invertedand placed into well 52 of instrument means 51 for measuring the reagentmeans response.

While the test device of the present invention is particularly suitedfor use in performing binding assays as discussed previously, variousother types of assay methods can be carried out using the test device.Conventional colorimetric assays may be carried out by selectingappropriate reagent means and appropriately orienting the reagentconstituents thereof and a sample receiving portion along the stripelement such that the desired color response is disposed at apredetermined measuring location upon completion of the test. In such anassay, the test device offers the advantage of accommodating sequentialtest reactions and has a high degree of accuracy because of the use of aprecisely dispensed aliquot of the test sample.

As a further example of the diverse types of assays that can beperformed using the test device of the present invention, there will nowbe described a test device for quantitatively determining proteins in afluid sample. Referring to FIGS. 3 and 4 of the drawing, strip element21 is provided with sample receiving portion 24 with portions 25 and 26being incorporated, respectively, with a label and with an agent forcoupling the label to proteins. As example of a label/coupling agentcombination is radioactive iodide and 1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril (sold as IODO-GEN by Pierce Chemical Co., Rockford, Ill.).The capillary advance of the developing fluid mixes the test sample andthe label, and when the resulting mixture enters portion 26, the labelis chemically coupled to proteins in the sample forming labeledderivatives. As the developing fluid advances farther, the labeledderivatives and free label are separated by their differing rates oftransport along strip element 21 by the developing fluid. Upon completetraversal of strip element 21 by the developing fluid, the labelresponse in the area of strip element 21 at which are disposed thelabeled derivatives provides a measure of the protein content of thesample. This type of test device can also be used to quantitativelydetermine a particular protein by positioning the coupling agentincorporated portion of the strip element a predetermined distance fromthe measuring location, i.e. positioning portion 26 a predetermineddistance from portion 27, where such predetermined distance correspondsto the distance that the labeled derivative of the protein of interestis transported during the time that the developing fluid travels fromthe coupling agent incorporated portion to the terminal end portion ofthe strip element, i.e. portion 28. The measuring point, i.e. portion27, will then contain the labeled derivative of the protein of interestonly.

The test device of the present invention can also be designed toquantitatively determine substances which react with a reagent system toproduce hydrogen peroxide. Referring to FIGS. 5-7 of the drawing, stripelement 31 is provided with sample receiving portion 34 and portions 35and 36 are incorporated, respectively, with radioactive iodide and withthe aforementioned reagent system. A preferred reagent system includesan oxidase enzyme specific for the substance to be determined. Forexample, to detect glucose or cholesterol, the reagent system wouldcomprise glucose oxidase or cholesterol oxidase, respectively. In use,the developing fluid mixes the sample and radioactive iodide. When themixture contacts the reagent system, hydrogen peroxide is produced inproportion to the amount of the substance to be determined present inthe sample with an attendant proportional conversion of radioactiveiodide to radioactive iodine, the latter being strongly adsorbed bystrip element 31. As the developing fluid advances, the remainingradioactive iodide is separated from the adsorbed radioactive iodine.The final location of either radioactive species upon completion of thetest can be the measuring location.

With reference to the particular embodiments of the test device of thepresent invention described hereinabove and in the Examples to follow,the present test device is applicable to the determination of acharacteristic of a sample and comprises (a) a strip element, usuallyelongated, having a beginning end portion and a terminal end portion andhaving a portion for receiving said sample positioned at a predeterminedlocation thereon, said strip element being composed of a materialcapable of transporting a developing fluid, usually longitudinally,therealong by capillarity, and (b) reagent means incorporated with saidstrip element at at least one predetermined location thereon forproviding a detectable response, as a function of the characteristicunder determination, at a predetermined measuring location on said stripelement upon traversal thereof by said developing fluid from saidbeginning end portion to said terminal end portion. The choice of thematerial composing the strip element, the composition of the developingfluid, the dimensions of the strip element, and the orientation of thesample receiving portion of the strip element and the portion orportions incorporated with the reagent means will, of course, dependupon the characteristic to be determined and the selected reagent means.

The strip element may be made of any material which is insoluble in thedeveloping fluid and which is capable of transporting the developingfluid by capillarity. The strip element typically is relatively flexiblewhile having a satisfactory wet strength to stand up under use. Ofcourse, it should be made of a material which will not deleteriouslyaffect the interactions between the developing fluid, the test sample,and the reagent means. A particularly useful material for the stripelement is bibulous paper, such as filter paper, since the developingfluid is usually aqueous in nature, however, other materials may also beused, including various felts, cloths, gels, membranes, and films madeof natural or synthetic substances including polymers. While the lengthand width of the strip element may vary widely, the thickness of thestrip element is usually between about 0.008 inch (0.2 mm) and 0.04 inch(4.0 mm).

The strip element is preferably affixed to an inert support formechanical strength. Usually, the strip element and inert support arejoined in laminate fashion with both being of approximately equal width.The thickness of the inert support may vary depending on the rigidity ofthe material of which it is made. Exemplary materials are the variousvinyl plastics as well as polyester, polycarbonate, methyl methacrylatepolymer, polystyrene, polyethylene, polypropylene, and waxed cardboard.The length of the inert support will vary depending upon the desiredconfiguration of the test device. The inert support may be approximatelythe same length as the strip element (as shown in FIGS. 3 and 4 of thedrawing) or, as is particularly preferred, may have a length greaterthan 0.5 times but less than 1.0 times the length of the strip elementin order that the transverse edge of the beginning end portion of thestrip element may be even with or slightly extended over one end of thesupport and the strip element folded widthwise over the other end of thesupport with the transverse edge of the terminal end portion of thestrip element being a spaced distance from the first mentioned end ofthe support (as shown in FIGS. 5-7 of the drawing). The latter mentionedpreferred configuration is most advantageous when the orientation of thesample receiving portion and the reagent means incorporated portion orportions are established such that the predetermined measuring locationis at or proximate to the fold in the strip element. This allowsconvenient measurement of the reagent means response at the measuringlocation by masking all but a minor portion of the strip elementproximate to the fold.

The terminal end portion of the strip element is preferably incorporatedwith indicator means responsive to the developing fluid to serve as asignal that the traversal of the strip element by the developing fluidis complete. For example, the indicator means may comprise acolorimetric reagent composition sensitive to a solvent or solute of thedeveloping fluid. Where the developing fluid is aqueous, the indicatormeans may contain a water sensitive reagent such as cobalt chloride and,in such case, the indicator means may also serve as a stabilityindicator. Also, the indicator means may comprise all of the componentsof a colorimetic reaction activated by the developing fluid. Forexample, where the developing fluid is aqueous, the indicator means mayinclude an acidic or basic material and a pH indicator.

The developing liquid must be capable of traversing the strip element bycapillarity and have solvent properties appropriate for the desiredcombination of the test sample and reagent means during such traversal.Usually, the developing solution is a solvent for appropriate substancesin the test sample, such as a substance to be detected and containsvarious ancillary agents such as stabilizing agents, preservatives, andinhibitory agents against interfering reactions.

Where a constituent of the reagent means is required to be incorporatedwith a portion of the strip element in an immobilized state, such may beaccomplished in any conventional manner. For example, the constituentmay be immobilized by physical adsorption or chemical coupling to thestrip element. An alternative method is to render the constituentinsoluble and immovable by the developing fluid such as by associatingit by physical or chemical means with a large insoluble particle. Forexample, where a binding assay test device is involved and one of thereagent constituents is a protein, such as an antibody, such can beimmobilized effectively by absorption to plastic beads.

In the case of a test device comprising reagent means that includes alabel, such as where a binding assay is to be performed, such label maybe any chemical substance or moiety having a detectable characteristicwhich is unique compared to the other materials involved in carrying outthe test. For instance, such label may have fluorescent ordistinguishable light absorption properties or reactivity. Particularlypreferred are substances which are radioactive because of the highdegree of sensitivity at which such can be detected. Radioactive labels,particularly radioactive iodine such as ¹²⁵ I and ¹³¹ I, areparticularly useful in carrying out binding assays using the test deviceof the present invention. While radioactive labels incorporated with aligand or a binding analog thereof elicit a measurable characteristicwhich is the same no matter whether such ligand or analog is bound to abinding partner or not, it is contemplated that labels may be used whichas incorporated with the ligand or analog yield characteristics whichare measurably different when the ligand or analog is in its bound statethan when it is in its free state.

As applied to binding assays, the present test device can be designed todetect any ligand for which there is a specific binding partner. Theligand usually is a peptide, protein, carbohydrate, glycoprotein,steroid, or other organic or inorganic molecule or ion for which aspecific binding partner exists in biological systems or can besynthesized. The ligand, in functional terms, is usually selected fromthe group consisting of antigens and antibodies thereto; haptens andantibodies thereto; and hormones, vitamins, metabolites andpharmacological agents, and their receptors and binding substances.Specific examples of ligands which may be detected using the presentinvention are hormones such as insulin, chorionic gonadotropin,thyroxine, triiodothyronine, estriol, testosterone, androsterone,equilenin, estrone, progesterone, pregnenolone, cortisol,17-hydroxydeoxy-corticosterone, and aldosterone, pharmacological agentsand their metabolites such as dilantin, digoxin, morphine, digitoxin,barbiturates, catecholamines, glutethimide, cocaine, diphenylhydantoin,meprobamate, benzdiazocycloheptanes, and phenothiazines; antigens andhaptens such as ferritin, bradykinin, prostoglandins, hemoglobin,enzymes, myoglogin, and tumor specific antigens, vitamins such asbiotin, the B vitamin group, vitamin A, the D vitamins, vitamins E andK, folic acid, and ascorbic acid; binding proteins such as antibodies,thyroxine binding globulin, avidin, intrinsic factor, andtranscobalamin; and other substances including antibodies, pesticides,fungicides, nematocides, living or non-living cells derived frombacteria, protozoa, fungi, viruses, and high order animals, and mineralssuch as calcium, ferrous and ferric ions, and oxalate, phosphate, andchloride ions.

In addition to the detection of particular substances of groups ofsubstances in a test sample, other characteristics may be determinedusing the present device. For example, ligand binding capacities may bedetermined where a ligand exists in the sample in a free form and abound form. The ligand binding capacity of the sample is the amount orpercent of exogenous free form ligand that is converted into the boundform when added to the sample (such as triiodothyronine binding capacityas involved in Example 2 hereof).

In most instances, the test reactions can be carried out successfully atroom temperature but, in general, the temperature at which the test isperformed may range from about 3° C. to about 45° C. with the reactionrate being generally directly related to the temperature.

The test sample may be a solid material but usually is a naturallyoccurring or artificially formed liquid suspected of containing or knownto contain the substance or characteristic to be determined. The presenttest device is particularly suited to assay biological fluids, ordilutions or other treatments thereof, such as serum, plasma, urine, andamniotic, cerebral, and spinal fluids. Solid matter, such as tissue orcells, or gases may be tested also by reducing them to a liquid formsuch as by dissolution of the solid or gas in a liquid or by liquidextraction of a solid.

The present invention will now be illustrated, but is not intended to belimited, by the following Examples.

EXAMPLE 1 Test for Thyroxine in Serum A. Preparation of Test Devices

A sheet of filter paper (Whatman #17 from W. & R. Balston Ltd.Maidstone, Kent, England) was cut into strips measuring 188 mm in lengthand 6 mm in width. Each paper strip was folded widthwise over one end ofa plastic strip 0.015 inch (0.38 mm) thick, 4 inches (102 mm) long, and1/4 inch (6.3 mm) wide, in such a fashion that one end of the paperstrip (designated the beginning end) was about even with one end of theplastic strip, leaving the other end of the paper strip (designated theterminal end) about 3/4 inch (19 mm) short of the same end of theplastic strip on the reverse side thereof. The folded paper strip wasaffixed to the plastic strip by means of the double-faced adhesive tape.

The following liquid mixtures were prepared:

Mixture A--A mixture of:

(1) 0.2 ml goat antiserum raised against a conjugate of thyroxine andhuman serum albumin (the antiserum was raised in a manner similar tothat described in J. Clin. Endo. 33: 509-16(1971) );

(2) 0.4 ml of an aqueous suspension of polystyrene beads containing 10%solids consisting of beads with an average diameter of 0.11μ (micron)(supplied by Sigma Chemical Co., St. Louis, MO);

(3) 2.4 ml of a 100 ml aqueous solution containing 694 mg potassiumdihydrogen phosphate, 509 mg disodium phosphate heptahydrate, 200 mgthimerosal (obtained from K&K Labs, Plainview, NY) and 0.5 mg crystalviolet;

(4) 0.2 ml of 5% aqueous sodium lauryl sulfate;

(5) 20 μl (microliters) of an aqueous solution containing 50 mggentamicin manufactured by Schering Corp., Bloomfield, NJ per ml; and

(6) a trace (about 1 μl) of silicone antifoam (AF 60 emulsion grademanufactured by General Electric and obtained from Harwick StandardChemical Co., Boston, MA).

Mixture B--A mixture of:

(1) 20 microcuries ¹²⁵ I-labeled thyroxine (obtained from CambridgeRadiopharmaceuticals Corp., Billerica, MA) in 0.2 ml of 50% propyleneglycol (specific activity of about 600 microcuries per mg);

(2) 3 ml of a 100 ml aqueous solution containing 5.32 g sodium barbital,1.44 ml 2 N hydrochloric acid, 400 mg thimerosal, 10 mg disodiumethylenediamine tetraacetate, and 0.5 mg crystal violet;

(3) 8 μl of an aqueous solution containing 50 mg gentamicin per ml; and

(4) 8 μl of an aqueous solution containing 250 mg human serum albuminper ml.

Mixture C--An aqueous solution containing 400 mg/100 ml thimerosal and10 mg/100 ml of red dye Ponceau S.

Mixture D--An aqueous solution containing 0.05 M hydrochloric acid and50 mg/100 ml bromocresol purple.

The above liquid mixtures were then applied dropwise to the paper stripsas follows:

    ______________________________________                                                             Point of application measured                            Mixture                                                                              Volume (μl)                                                                              from beginning end of strip (mm)                         ______________________________________                                        A      20            108                                                      B      20             80                                                      C      10             60                                                      D      10            185                                                      ______________________________________                                    

The strips were allowed to dry at room temperature.

A developing fluid was prepared to consist of 100 ml of an aqueoussolution containing 5.32 g sodium barbital, 1.44 ml 2 N hydrochloricacid, 400 mg thimerosal, and 10 mg disodium ethylenediaminetetraacetate.

Referring to FIGS. 5-7 of the drawing, the resulting test devices 30each included (a) sample receiving portion 34 (point of application ofMixture C) indicated by the dried spot of the red dye Ponceau S, (b)label incorporated portion 35 (point of application of Mixture B)including radiolabeled thyroxine, (c) specific binding partnerincorporated portion 36 (point of application of Mixture A) includingimmobilized antibody to thyroxine, and (d) terminal end portion 37(point of application of Mixture D) including, as indicator means, acombination of an acid and a pH indicator which produces a color changeupon contact by an alkaline liquid.

B. Assay Method

Ten (10) μl of a serum sample to be tested is applied to the samplereceiving portion 34 of a test device 30. The test device 30 is thenplaced, with the beginning end portion 33 down, in a test tube 41 (FIG.8) containing 1 ml of the developing solution. The size of the test tubeis selected so that the developing solution contacts the test device 30only at its beginning end portion 33, with no contact with its terminalend portion 37 (as depicted in FIG. 8). The test tube is capped andallowed to stand at room temperature until a color change from yellow toblue is observed at terminal end portion 37 (about one hour). At suchtime, all of the developing fluid in the test tube will have been drawnup into the strip element 31 of the test device 30. The test tube isthen inverted, all but 1/2 inch (12.2 mm) of the test device measuredfrom the fold of the paper strip is shrouded by inserting each tube intoa length of 5/8 inch (16 mm) O.D. copper tubing, and the tube is placedinto the counting well 52 (FIG. 9) of an In-V-Tron 200 gamma counter(manufactured by Nuclear Systems, Inc., Garland, Texas) to measure theamount of gamma radiation emitted at and proximate to the specificbinding partner portion 36 of the test device.

C. Principle of Test

As the developing fluid is transported up strip element 31 bycapillarity, the serum sample applied at portion 34 is encounteredfirst. The advancing developing fluid carries the serum sample,including any thyroxine present therein, to the label incorporatedportion 35. Endogenous non-radioactive thyroxine is mixed with labeledthyroxine as the developing fluid advances towards the fold of stripelement 31. The thyroxine mixture is carried over the fold in stripelement 31 by the developing fluid and then comes into contact withantiserum to thyroxine which is immobilized at portion 36. As thethyroxine mixture moves through portion 36, endogenous non-radioactivethyroxine and labeled thyroxine compete for antibody binding sites. Oncethe thyroxine mixture has passed through portion 36, the amount oflabeled thyroxine bound to immobilized antibody, and thereby itselfimmobilized at portion 36, is inversely related to the amount ofthyroxine present in the serum sample. Complete traversal of stripelement 31 by the developing fluid is indicated by a color change interminal end portion 37 which results upon wetting by the developingfluid.

D. Results

The assay procedure was run in duplicate on two (2) serum samples havingknown thyroxine contents (Thyroid Profile Control Sera from OxfordLaboratories, Inc., Foster City, CA). The results were as follows:

    ______________________________________                                        Type of Serum                                                                             Normal Control                                                                              Elevated Control                                    Control                                                                       Lot Number  14221         14222                                               Stated Thyroxine                                                                          6.2 ± 0.6 μg/100 ml                                                                   13.9 ± 0.7 μg/100 ml                          Content                                                                       Counts/min  13.71, 13.51  9.56, 9.70                                          (thousands)                                                                               av. 13.61     av. 9.63                                            ______________________________________                                    

These data indicate that the amount of labeled thyroxine resulting atand proximate to the fold of the test device after traversal of thestrip element by the developing fluid is an inverse function of theamount of thyroxine present in the serum sample tested.

EXAMPLE 2 Test for the Triiodothyronine Binding Capacity of Serum A.Preparation of Test Devices

Blank test devices were prepared by cutting paper strips and foldingthem over and affixing them to plastic strips in the manner described inPart A of Example 1.

The following liquid mixtures were prepared:

Mixture E--A mixture of:

(1) 9.12 g citric acid monohydrate,

(2) 14.12 g trisodium citrate dihydrate, and

(3) deionized water sufficient to make a total volume of 1 liter.

Mixture F--An aqueous solution containing 10 mg of red dye Ponceau S per100 ml.

Mixture G--A mixture of:

(1) 100 microcuries ¹²⁵ I-labeled triiodothyronine (obtained fromCambridge Radiopharmaceuticals Corp., Billerica, MA) in 1 ml of 50%propylene glycol;

(2) 20 ml of a 100 ml aqueous solution containing 5.32 sodium barbital,1.44 ml 2 N hydrochloric acid, and 1 mg crystal violet;

(3) 50 μl of an aqueous solution containing 50 mg gentamicin per ml; and

(4) 50 μl of an aqueous solution containing 250 mg human serum albuminper ml.

Mixture H--A mixture of:

(1) 50 mg hydroxypropyl guar gum (Jaguar HP-11 brand from Stein-HallSpecialty Chemical, New York, NY);

(2) 2 g microfine precipitated silica (QUSO 32 brand from PhiladelphiaQuartz Co., Valley Forge, Pa.);

(3) 0.1 ml of an aqueous solution of 500 mg crystal violet per 100 ml;and

(4) 150 ml of Mixture E.

Mixture J-An aqueous solution containing 0.02 M sodium hydroxide and 50mg/100 ml bromcresol purple.

The above liquid mixtures were then applied dropwise to the paper stripsas follows:

    ______________________________________                                                             Point of application measured                            Mixture                                                                              Volume (μl)                                                                              from beginning end of strip (mm)                         ______________________________________                                        F      10 μl       35                                                      G      10 μl       54                                                      H      30 μl      108                                                      J      10 μl      185                                                      ______________________________________                                    

The strips were allowed to dry at room temperature.

Mixture E was used as the developing fluid.

Referring to FIGS. 5-7 of the drawing, the resulting test devices 30each included (a) sample receiving portion 34 (point of application ofMixture F) indicated by the dried spot of the red dye Ponceau S, (b)label incorporated portion 35 (point of application of Mixture G)including radiolabeled triiodothyronine, (c) binding agent incorporatedportion 36 (point of application of Mixture H) including immobilizedsilica capable of adsorbing free triiodothyronine but nottriiodothyronine bound to serum proteins, and (d) terminal end portion37 (point of application of Mixture J) including, as indicator means, acombination of an alkali and a pH indicator.

B. Assay Method

The same procedure as described in Part B of Example 1 was followed.

C. Principle of Test

As the developing fluid is transported up strip element 31 bycapillarity, the serum sample applied at portion 34 is encounteredfirst. The advancing developing fluid carries the serum sample to thelabel incorporated portion 35 where it is mixed with labeledtriiodothyronine. The thyroxine binding globulin present in the serumsample binds an amount of the labeled triiodothyronine proportional tothe degree of its unsaturation. When the resulting mixture is passedthrough binding agent incorporated portion 36, free labeledtriiodothyronine, i.e. that not bound to thyroxine binding globulin, isadsorbed by the immobilized silica particles. Upon complete traversal ofstrip element 31 by the developing fluid, the amount of labeledtriiodothyronine immobilized at portion 36 is directly related to thepercent saturation of thyroxine binding globulin in the serum sample.

D. Results

The assay procedure was run in duplicate on two (2) serum samples havingknown triiodothyronine binding capacities (expressed as T-3 percentuptake) (control sera from Lederle Diagnostics, Pearl River, NY).

The results were as follows:

    ______________________________________                                        Type of Serum Control                                                                        "RIA Control I"                                                                            "RIA Control II"                                  Lot Number     2945-301     2946-301                                          Stated T-3 Percent Uptake                                                                    42.0         68.9                                              Counts/min (thousands)                                                                       10.35, 11.49 18.60, 18.02                                                     av. 10.92    av. 18.31                                         ______________________________________                                    

These data indicate that the amount of labeled triiodothyronineresulting at and proximate to the fold of the test device aftertraversal of the strip element by the developing fluid is a directfunction of the percent saturation of triiodothyronine binding proteinsin the serum sample tested.

EXAMPLE 3 Test for Folic Acid and Analogues Thereof in Serum A.Preparation of Test Devices

Blank test devices were prepared by cutting paper strips and foldingthem over and affixing them to plastic strips in the manner described inPart A of Example 1.

The following liquid mixtures were prepared:

Mixture K-A 150 ml aqueous solution containing 100 mg Baker gelatin(obtained from Doe and Ingalls Co., Medford, MA), 500 mg2-amino-2-(hydroxymethyl)-1, 3-propanediol, 100 mg ascorbic acid, 67 mgsodium azide and 0.383 ml 1 N sodium hydroxide.

Mixture L-A 20 ml volume of Mixture K containing 20 mgbeta-lactoglobulin (obtained from Sigma Chemical Co., St. Louis, MO).

Mixture M-A 0.5 ml volume of Mixture K containing 1.2 microcuries of ¹²⁵p-labeled folic acid (obtained from Diagnostic Biochemistry Inc., SanDiego, CA).

The above liquid mixtures and Mixture D from Example 1 were then applieddropwise to the paper strips as follows:

    ______________________________________                                                             Point of application measured                            Mixture                                                                              Volume (μl)                                                                              from beginning end of strip (mm)                         ______________________________________                                        L      20            92                                                       M      10            80                                                       D      10            185                                                      ______________________________________                                    

A light pencil mark was also made on each paper strip at a point 67 mmfrom the beginning end thereof. The strips were allowed to dry at roomtemperature.

Mixture K was used as the developing fluid.

Referring to FIGS. 8 and 9 of the drawing, the resulting test devices 30included (a) sample receiving portion 34 (indicated by the pencil mark),(b) label incorporated portion 35 (point of application of Mixture M)including radiolabeled folic acid, (c) specific binding partnerincorporated portion 36 (point of application of Mixture L) includingbeta-lactoglobulin, and (d) terminal end portion 37 (point ofapplication of Mixture D) including, as indicator means, a combinationof an acid and a pH indicator.

B. Assay Method

The same procedure as described in Part B of Example 1 was followed.

C. Principle of Test

As the developing fluid is transported up strip element 31 bycapillarity, it entrains first the serum sample and then theradiolabeled folic acid. The folic acid and its analogues present in thesample and the radiolabeled folic acid become mixed as the developingfluid advances to the binding partner incorporated portion 36.Beta-lactoglobulin has a limited ability to bind folic acid and itsanalogues and therefore as the sample/label mixture passes throughportion 36, a fraction of the amount of folic acid and its analogues inthe mixture become bound to beta-lactoglobulin. As the developing fluidadvances upward and over the fold in strip element 31, thebeta-lactoglobulin/folic acid or analogue complexes formed aretransported along strip element 31 but at a rate that is less than therate of transport of free folic acid and its analogues. Portions 36 ofstrip element 31 is positioned such that upon complete traversal ofstrip element 31 by the developing fluid, substantially all of thebeta-lactoglobulin/labeled folic acid complexes that have formed aredisposed at or proximate to the fold of strip element 31. Free labeledfolic acid meanwhile has been transported to the terminal end portion37. Therefore, the amount of radiolabeled folic acid resulting at andproximate to the fold of strip element 31 is inversely related to theamount of folic acid and its analogues in the serum sample.

D. Results

The assay procedure was run in duplicate on two (2) serum samples havingknown folate contents (control sera from Lederle Diagnostics, PearlRiver, NY). The results were as follows:

    ______________________________________                                        Type of Serum Control                                                                        "RIA Control I"                                                                            "RIA Control II"                                  Lot Number     2945-301     2946-301                                          Stated Folate Content                                                         (as determined by                                                             three different                                                               methods)                                                                      1st Method     4.0 mg/ml    3.8 mg/ml                                         2nd Method     3.6 mg/ml    2.9 mg/ml                                         3rd Method     2.5 mg/ml    2.2 mg/ml                                         Counts/min (thousands)                                                                       5.92, 6.06   8.49, 8.02                                                       av. 5.99     av. 8.26                                          ______________________________________                                    

These data indicate that the amount of labeled folic acid resulting atand proximate to the fold of the test device after traversal of thestrip element by the developing fluid is an indirect function of theamount of folate in the serum sample tested.

Obviously, many other modifications and variations of the invention asset forth hereinabove may be made without departing from the spirit andscope thereof.

What is claimed is:
 1. A test strip for determining a characteristic ofa sample, comprising a length of material capable of transporting adeveloping liquid therealong by capillarity, said strip includinga firstend, at which capillary transport begins; a second end, at whichcapillary transport ends; a plurality of zones positioned between saidfirst and second ends, said zones including a first zone for receivingsaid sample, a second zone impregnated with a first reagent capable ofbeing transported along said strip by said developing liquid, a thirdzone downstream of said first and second zones impregnated with a secondreagent, said first and second zones being spaced sufficiently from saidfirst end to permit contact of said first end but not said first andsecond zones with said developing liquid, said first reagent beingcapable of reacting with one of the following group consisting of (1)said sample, (2) said sample and said second reagent, and (3) saidsecond reagent in competition with said sample, to form a product in anamount dependent on said characteristic being determined, one of thegroup consisting of said second reagent and said material being aretarding element capable of slowing capillary transport of a movingelement, said moving element being one of the group consisting of saidproduct and said first reagent to thereby separate said product fromsaid first reagent, and said first reagent having a detectable propertythat provides a label for said first reagent and for said product topermit one of them to be a detectable agent whose amount is to bedetected; and a measuring location at or downstream of said third zone,said measuring location being spaced upstream of said second end adistance preselected so that, when transport of said developing liquidalong said strip element is terminated by the leading front thereofreaching said second end, said product and said first reagent have beenseparated sufficiently to permit the amount of said detectable agent tobe detected at said location by measuring said detectable property,whereby, after said sample is placed on said first zone and said firstend is dipped into said developing liquid, said developing liquidtransports said sample and first reagent to bring about the reaction toform said product, in an amount dependent on said characteristic; saidretarding element slows transport of said moving element to spatiallyseparate the two; and the amount of said moving element and thereby saidcharacteristic, is measured at said measurement location by measuringsaid detectable property.
 2. A test strip according to claim 1 furthercomprising an inert support to which said strip is affixed.
 3. A teststrip according to claim 2 wherein said support is a sheet laminated tosaid strip, the support sheet having a width about the same as that ofsaid strip and a length greater than one-half of, but less than thelength of said strip, the transverse edge of said first end being aboutco-extensive with one end of said support and said strip being foldedtransversely over the other end of said support so that the transverseedge of said second end is spaced from said first end.
 4. A test stripaccording to claim 3 wherein the third zone is at or proximate to thefold of said strip.
 5. A test strip according to any of preceding claims1-3 further provided with indicator means at said second end, responsiveto contact with said developing liquid for indicating when thedeveloping liquid has reached the second end.
 6. A test strip accordingto any one of claims 1 to 3 for detecting the binding capacity for afirst ligand of a sample which contains a second ligand in free andbound forms, wherein the first reagent includes a labelled form of thefirst ligand, and the second reagent comprises a binding agent capableof binding said labelled form of the first ligand when in one of saidfree form and said bound form but not when in the other, said labelledform and said binding agent being incorporated in said strip in theirrespective zones in proportions such that an amount of said product isproduced, which is a function of the ligand binding capacity of saidsample.
 7. A test device comprising a sealable chamber in which a teststrip according to any one of claims 1-3 is held upright therein withsaid first end of said length of material immersed in said developingligand.
 8. A test strip according to claim 1 for quantitativelydetermining a ligand ingredient in a sample, wherein said first reagentcomprises a labelled form of said ligand or of a specific binding analogthereof, the ligand and binding analog respectively being capable ofreacting in competition with the ligand in the sample, with a secondreagent which comprises a specific binding partner of said ligand, saidlabelled form and said binding partner being incorporated in said stripin their respective zones in proportions such that an amount of saidproduct is produced which is a function of the amount of said ligand insaid sample.
 9. A test strip according to claim 8 wherein said labelledform is a radioactive form of said ligand or of said binding analogrespectively.
 10. A test strip according to claim 1 for determiningquantitatively protein(s) in a liquid sample, wherein said secondreagent comprises an agent for coupling said first reagent to protein(s)to provide labelled proteins.
 11. A test strip according to claim 10 fordetermining a particular protein quantitatively, in which said measuringlocation is positioned to measure the amount of coupled producttransported by said developing liquid when said developing liquidreaches said second end.
 12. A test strip according to claim 10 or 11wherein said first reagent is radioactive iodide.
 13. A test stripaccording to claim 1 for quantitatively determining in a liquid sample afirst substance which reacts with a second substance to produce hydrogenperoxide wherein said first reagent comprises radioactive iodide andsaid second reagent comprises said second substance.
 14. A test stripaccording to claim 13 for quantitatively determining in a liquid samplea substrate for an oxidase enzyme reaction which produces hydrogenperoxide, wherein said second substance comprises said oxidase enzyme.15. The test strip of claim 1 wherein said detectable property isradioactivity.
 16. The test strip of claim 1 whereinsaid first reagentis selected to react with said sample to form an amount of said productthat is dependent on said characteristic and said second reagent isselected to slow transport of the portion said first reagent unreactedwith said sample.
 17. The test strip of claim 1 whereinsaid firstreagent is selected to be mixable with said sample and transportabletherewith to said third zone and said second reagent is selected toreact in said third zone with at least said sample to form said product.18. The test strip of claim 17 whereinsaid second reagent reacts withsaid sample and said first reagent in said third zone and said firstreagent is selected to be sufficiently chemically similar to aningredient of said sample as to complete with said ingredient inreacting with said second reagent to form said product, whereby theamount of said product is dependent on the amount of ingredientcompeting therewith in reacting with said second reagent.
 19. The teststrip of claim 18 wherein said second reagent slows transport of saidproduct.
 20. The test strip of claim 17 whereinsaid second reagentreacts with said sample and said first reagent to form said product. 21.The test strip of claim 20 wherein transport of said product is slowedby said material.